Simple Extraction Cum RP-HPLC Method for Estimation of Nanotized Quercetin in Serum and Tissues of Mice

In recent years, several studies have focused on antioxidant, anti-inflammatory, and anti-cancer activities of quercetin (3,3',4',5, -pentahydroxyflavone). The nanotization of quercetin was shown to enhance its therapeutic efficacy due to smaller particle size. In the present study, an additional step was added to simple extraction cum RP-HPLC method for the quantification of nanotized quercetin (nQ) in biological samples to understand the pharmacokinetics and biodistribution of nQ following intravenous administration. The proposed method involves extraction of nQ from blood serum and tissues of mice with 2N HCl in comparison to well-known DMSO:MeOH mix method. The HCl extraction was found to be 2 - 3 times more efficient than DMSO:MeOH mix method. Results showed that the amount of nQ at various time intervals in the serum and tissues was 2 - 3 fold greater for HCl extraction than for DMSO:MeOH mix method, suggesting that HCl extraction must take into account nQ bound with protein. The reversed-phase HPLC was used for nQ detection, which showed the nQ retention time of 3.2 min. The limit of detection of nQ in blood serum was found to be 0.1 mu g/mL. The proposed method was also validated in terms of linearity, precision, and accurac

Correction: Topical Application of Ochratoxin A Causes DNA Damage and Tumor Initiation in Mouse Skin.

[This corrects the article DOI: 10.1371/journal.pone.0047280.]

Topical application of ochratoxin A causes DNA damage and tumor initiation in mouse skin.

Skin cancer is one of the most common forms of cancer and 2-3 million new cases are being diagnosed globally each year. Along with UV rays, environmental pollutants/chemicals including mycotoxins, contaminants of various foods and feed stuffs, could be one of the aetiological factors of skin cancer. In the present study, we evaluated the DNA damaging potential and dermal carcinogenicity of a mycotoxin, ochratoxin A (OTA), with the rationale that dermal exposure to OTA in workers may occur during their involvement in pre and post harvest stages of agriculture. A single topical application of OTA (20-80 µg/mouse) resulted in significant DNA damage along with elevated γ-H2AX level in skin. Alteration in oxidative stress markers such as lipid peroxidation, protein carbonyl, glutathione content and antioxidant enzymes was observed in a dose (20-80 µg/mouse) and time-dependent (12-72 h) manner. The oxidative stress was further emphasized by the suppression of Nrf2 translocation to nucleus following a single topical application of OTA (80 µg/mouse) after 24 h. OTA (80 µg/mouse) application for 12-72 h caused significant enhancement in- (a) reactive oxygen species generation, (b) activation of ERK1/2, p38 and JNK MAPKs, (c) cell cycle arrest at G0/G1 phase (37-67%), (d) induction of apoptosis (2.0-11.0 fold), (e) expression of p53, p21/waf1, (f) Bax/Bcl-2 ratio, (g) cytochrome c level, (h) activities of caspase 9 (1.2-1.8 fold) and 3 (1.7-2.2 fold) as well as poly ADP ribose polymerase cleavage. In a two-stage mouse skin tumorigenesis protocol, it was observed that a single topical application of OTA (80 µg/mouse) followed by twice weekly application of 12-O-tetradecanoylphorbol-13-acetate for 24 week leads to tumor formation. These results suggest that OTA has skin tumor initiating property which may be related to oxidative stress, MAPKs signaling and DNA damage

Effect of topical application of OTA on oxidative stress in mouse skin.

<p>(A) Dose dependent effect of OTA on epidermal LPO, Protein Carbonyl, GSH content, and activities of Catalase, GST and GR in mouse skin exposed for 24 h. (B) Time dependent effect of OTA (80 µg/mouse) on epidermal LPO, Protein Carbonyl, GSH content, and activities of Catalase, GST and GR in mouse skin exposed for 12–72 h. (C) Time dependent effect of OTA (80 µg/mouse) on intracellular ROS generation in mouse skin exposed for 12–72 h. Single cell suspension from vehicle or OTA (80 µg/mouse) treated mouse skin was prepared and 1×10⁵ cells were incubated with 100 µM H₂DCFDA for 90 min at 37°C and relative fluorescence intensity was determined by spectrofluorimeter (λ_ex =485 nm, λ_em =520 nm). Data are represented in terms of fluorescence intensity (arbitrary units). Each value represents mean ± S.E. of five animals. *p<0.05, significant with respect to control group.</p

Effect of topical application of OTA on the translocation of Nrf2 transcription factor and phosphorylation of MAPKs proteins in mouse skin.

<p>(A) Nuclear extract was prepared from vehicle or OTA (80 µg/mouse) treated mouse skin and nuclear Nrf2 level was analysed by western blot analysis. Values above the lanes of blots are mentioned as fold change with respect to control. (B) Whole cell extract was prepared from vehicle or OTA (80 µg/mouse) treated mouse skin and p-ERK1/2 (Thr²⁰²/Tyr²⁰⁴), p-p38 (Thr¹⁸⁰/Tyr¹⁸²) and p-JNK (Thr¹⁸³/Tyr¹⁸⁵) levels and their respective total forms were analysed by western blot analysis. The values mentioned on each lane of blot represent the fold change of ratio of phosphorylated/total form with respect to control. For confirmation of equal protein loading, the blots were stripped and probed with an antibody specific for either tubulin or β-actin.</p

Different cell cycle phases of epidermal cells of mice topically treated with OTA (80 µg/mouse).

<p>Data represent mean ± SE of five animals. Details of treatment schedule and processing of cells are described in the Materials and Methods.</p><p>Values in parenthesis indicate percent increase (↑) or decrease (↓).</p>*<p>p<0.05, significant when compared with control.</p

Effect of topical application of OTA on apoptotic biochemical parameters in mouse skin.

<p>(A) Whole cell extract was prepared from vehicle or OTA (80 µg/mouse) treated mouse skin and p53 and p21/waf1 levels were analysed by western blot analysis. Values above the lanes of blots are mentioned as fold change with respect to control. (B) Bax, Bcl-2 and cytochrome <i>c</i> (cyt <i>c</i>) levels in mouse skin following OTA (80 µg/mouse) exposure were analysed by western blot analysis. Values above the lanes of blots are mentioned as fold change with respect to control. (C) Bax/Bcl-2 ratio calculated on the basis of densitometric analysis of respective western blots shown in (B), (D) Caspase 3, 8 and 9 activity in mouse skin treated with OTA (80 µg/mouse) was measured using specific substrate in terms of relative fluorescence unit, at an excitation and emission wavelengths of 400 and 505 nm, respectively. Data represents mean ± S.E. of five animals and depicted as fold change as compared to control. *p<0.05, significant with respect to control, (E) Cleavage of PARP following OTA (80 µg/mouse) exposure was analysed by western blot analysis. For confirmation of equal protein loading, the blots were stripped and probed with an antibody specific for β-actin.</p